Isolation of iPSC-Derived Cardiomyocytes

To sort the iPSC-aCMs from the CFs for CM-specific end-point analysis, the cells were disassociated as described above. Once the cells were in suspension, the PSC-Derived Cardiomyocyte Isolation Kit (Miltenyi Biotec, Gaithersburg, MD) was used to isolate the CMs via negative selection per the manufacturer’s protocol with LS columns and a MidiMACS Separator magnet (Miltenyi Biotec). Only labeling of the non-CM population was performed to reduce handling of the iPSC-aCMs. This has been shown previously to be sufficient for CM isolation (67 (link)) and, in our hands, yielded ~80 to 90% pure iPSC-aCMs and <6% fibroblasts based on cTnT and collagen 1a1 (Col1a1) immunostaining analysis, respectively (fig. S16). CM cell sorting was performed before RT-qPCR, RNA-seq, patch clamping, and Seahorse analysis.